CN103923067B - The micromolecular inhibitor of a kind of MdmX/Mdm2 and preparation method and application - Google Patents
The micromolecular inhibitor of a kind of MdmX/Mdm2 and preparation method and application Download PDFInfo
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- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
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Abstract
The present invention relates to field of pharmaceutical chemistry technology, specifically disclose the micromolecular inhibitor of a kind of MdmX/Mdm2, also relate to the preparation method of the micromolecular inhibitor of a kind of MdmX/Mdm2, this molecule inhibitor compounds can suppress MdmX protein and the interaction of p53 albumen, also Mdm2 protein and the interaction of p53 albumen can be suppressed, this molecule inhibitor compounds has antiproliferative activity to cancerous cell, and this molecule inhibitor compounds will not produce toxic and side effects to patient.This molecule inhibitor compounds can be used in combination with other therapies.
Description
Technical field
The present invention relates to field of pharmaceutical chemistry technology, the little molecule being more particularly to a kind of MdmX/Mdm2 presses down
Preparation, also relates to the preparation method of the micromolecular inhibitor of a kind of MdmX/Mdm2, and this little molecule presses down
Inhibitor compound can suppress Mdmx protein and the interaction of p53 albumen, also can suppress Mdm2 albumen
Matter and the interaction of p53 albumen, this molecule inhibitor compounds has antiproliferative activity to cancerous cell.
Background technology
The inactivation of p53 albumen function of tumor inhibition is relevant to the generation of most of human tumors.Muller etc. have rated
P53 gene mutation or suppressed by Mdm2 and MdmX albumen, is inactivation and the tumor generation phase of p53 function
Close mechanism (Muller, P.A.&Vousden, K.H.Nat.CellBiol.15,2-8 (2013).Wade etc. analyze
Find increasing evidence show Mdm2 and/or MdmX expression raise account for cancer nearly half (Wade, M.,
Li,Y.C.&Wahl,G.M.Nat.Rev.Cancer13,83-96(2013).Cheok etc. point out in expression wild
In the tumor of type p53, by suppression Mdm2 and MdmX, release activity p53 albumen, reach to eliminate tumor and make
With, be cancer drug design one of focus (Cheok, C.F., Verma, C.S., Baselga, J.&Lane,
D.P.Nat.Rev.Clin.Oncol.8,25-37,2011).
In the cancerous cell of Mdm2 gene amplification, Mdm2 inhibitor has shown that and promotes the thin of p53 mediation
Born of the same parents' cycle arrest, apoptosis and tumor regression.One of the most promising candidate, Russell Co., Ltd of the U.S.
The Mdm2 inhibitor nutlin-3a of invention is carrying out clinical research.But, owing to cancer uses other machines
System weakens or disables p53 signal, and the process LAN of such as another kind of p53 negative regulator MdmX can suppress
P53 protein active.The research of Macchiarulo, A etc. shows, in the tumor cell expressing wild type p53,
Suppression Mdm2 and MdmX can significantly more activate p53 base than the medicine of only suppression Mdm2 activity
Because of (Macchiarulo, A., Giacche, N., Carotti, A., Moretti, F.&Pellicciari, R.Med.Chem.
Commun.2,455-465,2011).In order to overcome the p53 protein active of tumor cell to lose,
Popowicz, G.M. etc. propose exploitation MdmX inhibitor and Mdm2/MdmX bispecific inhibitor has and faces
Bed meaning (Popowicz, G.M., Domling, A.&Holak, T.A.The structure-based design of
Mdm2/Mdmx-p53inhibitors gets serious.Angew.Chem.Int.EdEngl.50,2680-2688
2011)。
The micromolecular inhibitor structure of the Mdm2 having now been found that has following a few class, 1), cis-imidazolines.
Cis-imidazolines (the Chinese patent as MDM2 inhibitor has been invented by Hoffman-Laluoai Ltd
Application number: 02825229.2), Hoffman-Laluoai Ltd have invented successively modified model cis-imidazoline
The MDM2 inhibitor (Chinese Patent Application No.: 200480016900.X) of class, 2,4,5-triphenylimidazolyl quinoline
Derivant (Chinese Patent Application No.: 200680044810.0) and cis-4,5-diaryl-2-heterocycle-imidazoline (in
State's number of patent application: 200780002250.7).Hoffman-Laluoai Ltd also invention piperidines Mdm2
Inhibitor (Chinese Patent Application No.: 200480006687.4), Amgen Inc has invented piperidones and has spread out
Biological Mdm2 inhibitor (Chinese Patent Application No.: 201180038476.9), Janssen Pharmaceutica N. V is sent out
Understand cyclic-alkylaminederivativeas (Chinese Patent Application No.: 200780010151.3), pyrrolidines
(Chinese Patent Application No.: 201080046174.1), quaternary heteroaryl chemical combination has been invented by Novannis company
Thing (number of patent application: 201080048250.2), spiro-oxindole (Chinese patent application has been invented by University of Michigan
Number: 201080061294.9), MSU also invented Spiro-oxindole (Chinese Patent Application No.:
201280033358.3).These micromolecular inhibitors are strong to Mdm2 targeting, and affinity is high.Through structure
Compare with activity relationship, the structure of functional group and the combination feature of protein surface in these compounds, and multiple official
The same effect combined can be rolled into a ball and determine the affinity of little molecule and albumen.In above-mentioned patent application, quaternary
Heteroaryl compound patent (number of patent application: 201080048250.2) declares its compound to Mdm4 (i.e.
MdmX) there is effect, but its affinity is relatively low.Before making the present invention, there are no based on cis-imidazoline frame
The high-affinity MdmX reversible inhibitor patent report of frame, the height that there are no cis-imidazoline framework is affine
Power Mdm2/MdmX double inhibitors patent report, also there are no with cis-imidazolines and indole combined
High-affinity MdmX inhibitor and Mdm2/MdmX double inhibitors report.
Chang etc. confirmed respectively Mdm2 by little molecule double inhibitors by stable polypeptide and Graves etc. and
The inhibitory action that MdmX expresses can overcome tumor growth and decline (Chang, the Y.S.etal. that process LAN MdmX causes
Proc.Natl.Acad.Sci.U.S.A110, E3445-E3454,2013；Graves,B.etal.Proc.Natl.
Acad.Sci.U.S.A109,11788-11793,2012).Micromolecular inhibitor involved in the present invention is many
Planting display in cancer cell model and have wild type p53 dependency activity, its Anticancer Effect and Mechanism comes from activation
P53 approach.Present invention demonstrates that Mdm2/MdmX reactivates p53, inducing cell apoptosis is to control by suppression
Treat the available strategy of cancer.
Summary of the invention
For the deficiencies in the prior art, it is an object of the invention to there are provided a kind of MdmX/
The micromolecular inhibitor of Mdm2, described inhibitor can with p53 albumen-MdmX albumen in anticancer it
Between interaction, release activity p53 albumen, induction cancerous cell self-apoptotic process.Described MdmX
/ Mdm2 can also interaction between p53 albumen-Mdm2 albumen in anticancer, release further
The property let live p53 albumen, the self-apoptotic process of strengthening induction cancerous cell.
The micromolecular inhibitor of above-mentioned MdmX/Mdm2, its chemical structural formula is as follows:
Wherein, R1For phenyl C6H5, 2,4-Dichlorobenzene base-(2,4)-C6H3Cl2, 4-hydroxy phenyl-4-OH-C6H4、
4-carboxyl phenyl-4-C6H4-COOH or 4-Methanamide phenyl-4-C6H4-CONH2。
R2It is 3,5-Dichlorobenzene base-(3,5)-Cl-C6H3, 3,4,5-trichlorophenyl-(3,4,5)-Cl-C6H2、
2,4,6-trichlorophenyl-(2,4,6)-Cl-C6H2, 4-chloro-indole base-(4-Cl)-indole or indyl.
R3For 2-(1,1,1-trimethyl) methoxyl group-4-methoxyphenyl-C6H3-2-OC(CH3)3-4-OCH3、
2-(1,1,1-trimethyl) methoxyl group-4-cyano-phenyl-C6H3-2-OC(CH3)3-4-CN, 2-(1,1,1-
Trimethyl) methoxyl group-4-(1-acetic acid) methoxyphenyl-C6H3-2-OC(CH3)3-4-OCH2-CH2COOH、
2-(1,1,1-trimethyl) methoxyl group-4-(1-acetamide) methoxyphenyl
-C6H3-2-OC(CH3)3-4-OC-CH2CONH2Or 2-(1,1,1-trifluoromethyl) methoxyl group-4-(1-second
Amide) methoxyphenyl-C6H3-2-OC(CF3)3-4-CO-CH2CONH2。
Above-mentioned three sections of contents lisies are expressed as follows:
In table, the second row functional group is followed successively by R1(2)、R1(3)、R1(4)、R1(5)、R1(6)；
The third line functional group is followed successively by R2(2)、R2(3)、R2(4)、R2(5)、R2(6)；
Fourth line functional group is followed successively by R3(2)、R3(3)、R3(4)、R3(5)、R3(6)；
Another object of the present invention is to there are provided the micromolecular inhibitor of a kind of above-mentioned MdmX/Mdm2
Preparation method, easy to implement the method, easy and simple to handle, by have diastereomeric and stereo selectivity catalyst virtue
Base nitromethane and through the additive reaction of schiff bases.This is synthesized cis-imidazolines efficiency height,
Fewer than other synthetic method path (Davis, T.A.&Johnston, J.N.Chem.Sci.2,1076-1079,
2011).Basic synthesis step is shown in Fig. 1.
Wherein, functional group R1Use bromo halogeno-benzene alkane be raw material, by Kornblum describe method prepare (JACS,
1956,78:1497).Functional group R1(4) hydroxyl, R1(5) carboxyl and R1(6) amide is respectively adopted methyl
Ether, methyl ester and benzyl protection, deprotect post-synthetic phase respectively.Functional group R2Use a-aminobenzene sulfone or a-ammonia
Base indole sulfone is Material synthesis (Marianacci et.al., Chem-Euro J, 2007,13:8338).Utilize carbon
Acid potassium effect carries out eliminating reaction and produces schiff bases.Functional group R3Using corresponding benzoic acid is raw material, functional group
R3(4) carboxyl and R3And R (5)3(6) amide is respectively adopted methyl ester and benzyl protection, and post-synthetic phase is respectively
Deprotection.
In conjunction with Fig. 1, by synthesis step, details are as follows:
First, the aryl imine (compound 7 in Fig. 1) of tertbutyloxycarbonyl protection and nitro-phenyl alkyl (in Fig. 1
Compound 8) be dissolved in toluene (0.1M) by 1:1.1 equivalent, trifluoromethayl sulfonic acid (HOTf, 5% mole
Than) under catalytic action, within 19～27 hours, generated in the middle of beta-amino nitroparaffin by additive reaction-78 DEG C of reactions
Body (compound 9 in Fig. 1).Thereafter, utilize boronation cobalt that nitro in compound 9 is reduced into amino, it is provided that
Acylation reaction introduces R3Functional group, generates the amide compound (compound 10 in Fig. 1) of tertbutyloxycarbonyl protection.
Finally, directly going t-butoxycarbonyl protecting group to generate secondary amine with TFA, productivity is more than 90%, then through N,
Under N-carbonyl dimidazoles catalyst, amido acylation reaction generates Carbimide. intermediate product, and this intermediate product is through piperazinones
Processing and generate cis-imidazolines nutlin-3a analog, whole productivity is 71～89%.
Utilize LC-MS monitoring reaction course, until converting completely.Reactant use water extracts, and water layer is again
Extract with dichloromethane.Organic layer is merged, is dried with sodium sulfate, concentrate, obtain crude product.Utilize Waters
Reversed-phase HPLC (C18 post is further purified, and flow phase: containing the water of 0.1% formic acid, and containing 0.1% formic acid
Methanol, and further by SFC (OD-H post) separate to obtain pure enantiomer.
A further object of the invention is the little molecules in inhibiting that there are provided a kind of above-mentioned MdmX/Mdm2
Agent application in the self-apoptosis of induction cancerous cell, the especially application in treatment tumor.Clinical research shows
Showing, in many cancers, p53 albumen is still wild type, but is suppressed by MdmX and Mdm2 overexpression.
Hepatocarcinoma including 46%, the pulmonary carcinoma of 18%, the neuroendocrine tumour of 57%, the retinoblastoma of 65%,
The G. cephalantha of 50%, the breast carcinoma of 31%, 49% rectal cancer etc. all relevant to MdmX process LAN.Separately
Outward, the lipoma of 70%, the skin carcinoma of 37%, breast carcinoma of 31% etc. is relevant to Mdm2 process LAN.This
The micromolecular inhibitor of bright MdmX/Mdm2 has effect to above-mentioned cancer.
The compound of the present invention demonstrates the interaction of suppression MdmX and Mdm2 albumen and p53-sample peptide,
Its effect is higher than p53-derived peptide more than 200 times.In cell experiment, the compound of the present invention has shown that
Active mechanism.Cancerous cell is incubated together with wild type p53 and causes p53 protein accumulation, induction p53-to regulate
P21 gene and cell cycle arrest are in G1 and the G2 phase.Which results in vitro to wild type p53 cell
Antiproliferative activity.On the contrary, under suitable compound concentration, do not have in the cancerous cell containing mutant p53
Observe these activity.Therefore, the activity of MdmX inhibitor may function machine-processed relevant.These are changed
Compound can be effective and selective anticarcinogen.
In order to realize above-mentioned purpose, the present invention uses techniques below measure:
The present invention is prepared in Mdm2 and MdmX with p53 albumen combined structure territory for micromolecular inhibitor
Screening, affinity measures, little molecule and target point protein composite structure measure.People source MdmX aminoacid
Sequence 22-110 (N-MDMX) and people source Mdm2 aminoacid sequence 22-110 (N-Mdm2) DNA use
Colibacillus engineering is expressed, prepared by purification, is used for carrying out high flux inhibitor screening, ITC experiment and nuclear-magnetism
Resonance laboratory.
Recombiant protein is prepared in e. coli bl21 (DE3) cell.Cell cultivation is carried out with LB culture medium,
At 20 DEG C, 12 hours expressing proteins are induced with the IPTG of 0.4mM.Cell culture is centrifuged 8000 × g30
Minute, and be resuspended in lysis buffer, carry out supersound process.Lysate is centrifuged by 100,000 × g
Clear cell debris, and supernatant is loaded to the Ni-NTA agarose column (Qiagen) of 20ml column volume
Purification.Through 4 DEG C of digested overnight of TEV protease, remove the His label of protein, be then passed through second time
Ni-NTA agarose chromatography purification.Eluted protein component is purified with S200 solvent resistant column further.
Peak part merges, and is concentrated into 1 mg/ml then with liquid nitrogen flash freezer, and be stored in-80 DEG C standby.
Nuclear magnetic resonance experiment uses 15N labelling, containing BME vitamin (Sigma) with protein example is unified
M9 minimal medium in, with 1 grams per liter 15N-(NH4)2SO4With the glucose of 1 grams per liter respectively as
Nitrogen and the exclusive source of carbon.
The present invention utilizes nuclear magnetic resonance measuring to compare to there is nutlin-3a to N-MdmX's and N-Mdm2
The impact of H1-N15HSQC nuclear magnetic resonance map and compare and there is nutlin-3a to N-MdmX and N-Mdm2
The impact of structure dynamics.
The present invention uses fluorescence polarization (FP) method to carry out high flux MdmX inhibitor screening.Use fluorescein
It is marked p53 peptide inhibitor replacement protein/nutlin analog complex small molecular principle and measures little molecule
Affinity (IC50).P53p peptide behaviour source aminoacid sequence number 15-29, its aminoterminal of fluorescein labelling is (glimmering
Light element-GSGSSQETFSDLWKLLPEN, Flu-p53p).Its mutant p53 peptide (fluorescein
-GSGSSQETASDLAKLAPEN, Flu-p53pAAA) it is used as negative control.Utilize unlabelled many
Peptide and nutlin-3a make positive control.In library, MdmX guide's inhibitor screening standard controls IC50Value is less than
300nM.Present invention discover that the IC of six kinds of compounds50Value is respectively less than 300nM.
The present invention measures above-mentioned six kinds of guide's inhibitor pair further with the micro-calorimeter of isothermal titration (ITC)
N-MdmX and N-MdmX protein affinity (Kd).Binding constant Kd(both 1/Ka) it is that solution is by saturated
The slope of the centre straight ahead part of curve determines, Δ H obtains from ITC titration curve, Gibbs free energy Δ G
Calculated by Δ G=-RTlnKa=Δ H-T Δ S formula respectively with Entropy Changes Δ S.
Measuring through ITC and analyze, Mdm2 is suppressed by the six kinds of MdmX guide's inhibitor filtered out from library
Effect is also apparent from (table 1).Wherein, in a kind of lead compound, i.e. following table compound H109 to N-MdmX
With N-Mdm2 two, there is higher affinity, with the K of N-MdmX and N-Mdm2dValue is respectively 2.7nM
With 5.7nM (table 1).The ITC thermodynamics collection of illustrative plates of compound H109 titration N-MdmX and N-Mdm2
As shown in Figure 6.
Table 1
The micromolecular inhibitor of part MdmX/Mdm2 of the present invention is containing wild-type p 53 protein
Cancerous cell can block the MdmX inhibitory action to p53, it is possible to suppression MdmX and Mdm2 pair simultaneously
The inhibitory action of p53.Present invention discover that compound H109 can stop MdmX/Mdm2-p53 to interact.
This new nutlin analog should be only effective in the cell containing wild type p53, and is dashing forward containing transcriptional inactivation
Become p53 cell in the most invalid.Many cancers, p53 albumen is still wild type, but by MdmX and Mdm2
The suppression of overexpression.The hepatocarcinoma of such as 46%, the pulmonary carcinoma of 18%, the neuroendocrine tumour of 57%, 65%
Retinoblastoma, the G. cephalantha of 50%, the breast carcinoma of 31%, the rectal cancer etc. of 49% and MdmX
Process LAN is correlated with.It addition, the lipoma of 70%, the skin carcinoma of 37%, breast carcinoma of 31% etc. and Mdm2 mistake
Express relevant.Therefore, the micromolecular inhibitor of the present invention in above-mentioned treatment of cancer by powerful.Present invention profit
With the tumor cell line containing wild type p53, including colorectal cancer cell HCT116 and RKO and lung carcinoma cell
H460a is model, evaluates compound H109 micromolecular inhibitor anticancer activity.
Above-mentioned 3 kinds of tumor cell lines, after compound H109 processes 8 hours, pass through real-time polymerase chain reaction
(RT-PCR) expression of p53 and p21 gene is monitored.In all cells system, transcribing of p21 gene, with
The result of positive control nutlin-3a is consistent, all increases in the way of dependent dose.By contrast, p53 gene
Transcribing of itself is not affected by compound H109 process.After present invention discover that compound H109 is by translation
Mechanism raises p53 vigor, similar to positive control nutlin-3a effect.
Present invention mtt assay analysis of compounds H109 to the targeting of cell and grows viability inhibitory action.
Mouse model is utilized to prepare Mdm2 deficiency, MdmX deficiency lung cancer in mice embryo fibroblast (MEF).
With nutlin-3a as positive control and nutlin-3b as negative control, non-totally ordered MEF cell is through chemical combination
After thing H109 processes 48 hours, vigor substantially reduces, the same with positive control nutlin-3a, presents dosage and depends on
Lai Xing.MdmX deficiency MEF cell also presents dose dependent after compound H109 processes 48 hours
Change.It is essential that Mdm2 deficiency MEF cell presents dosage after compound H109 processes equally
Dependent change.On the contrary, the double defect MEF cell of Mdm2 and MdmX is not the most by compound
The impact of H109.It is strictly to rely on p53 that nutlin analog institute of the present invention mediated cell viability reduces
Vigor.
The present invention is calculated by molecular model and finds that the p53p peptide on compound H109 and N-MdmX surface is combined
Site is the most identical.
Compared with prior art, advantages of the present invention and having the beneficial effects that:
1, existing micromolecular inhibitor can only suppress the interaction of p53 and Mdm2, the little molecules in inhibiting of the present invention
Agent can suppress the interaction of p53 and MdmX and Mdm2 simultaneously, it is adaptable to further types of cancer.
2, many treatments of cancer usually can cause serious side effect.This micromolecular inhibitor cancer drug, special
Opposite sex ground for the interaction of p53 and MdmX and Mdm2 without patient is produced toxic and side effects.
3, this micromolecular inhibitor affinity is high, anticancer by force, more have adaptability than peptide inhibitor.
4, this micromolecular inhibitor can and other anti-cancer therapies be used in combination, effect is more preferable.
5, this micromolecular inhibitor is with sick cell as target spot, can efficiently and optionally killing tumor cell,
Reduce the damage of normal tissue, particularly overcome classic chemotherapy medicine poor specificity, shortcoming that toxic and side effects is big.
6, molecular target cancer therapy drug is conducive to a lot of tumors being become chronic process, as diabetes, heart disease etc.
Common chronic disease is the same, can preferably lead cancer patient to stride forward to " band tumor Centrum ".
Accompanying drawing explanation
Fig. 1 is the micromolecular inhibitor synthetic route schematic diagram of a kind of MdmX/Mdm2 of the present invention.
Fig. 2 is a kind of nutlin-3a analog library schematic diagram, and each functional groups number is from nutlin-3a
Counting.
Fig. 3 is that one compares when there is nutlin-3a, the H of N-MdmX and N-Mdm21-N15HSQC nuclear-magnetism
Resonance collection of illustrative plates.
Fig. 4 is that one compares when there is nutlin-3a, { the H of N-MdmX and N-Mdm2 amino acid residue1}-N15
Heteronuclear nOe diversity schematic diagram: on (), N-MdmX Yu the nutlin-3a mixture (1:1 of 15N labelling
Mol ratio) sample；In (), the secondary structure of N-Mdm2 and N-MdmX albumen, rectangle: α-helixstructure,
Arrow: beta sheet structure, fine rule: disordered structure；Under (), N-Mdm2 and nutlin-3a of 15N labelling
Mixture (1:1 mol ratio) sample.
Fig. 5 is a kind of with N-MdmX for target spot employing high throughput method screening guide's inhibitor.Change from design
In compound library, find six kinds of compounds IC to N-MdmX50Value is respectively less than 300nM.
Fig. 6 is the ITC thermodynamics of a kind of compound H109 titration N-MdmX (left) andN-Mdm2 (right)
Collection of illustrative plates.
Fig. 7 is nutlin-3a and the nutlin-3b structural formula mentioned in a kind of present invention.
Fig. 8 is that a kind of comparative compound H109 induces cancerous cell HCT116, RKO and H460a Han wild type p53
Middle p21 and p53 gene expression.Real circle symbol: p21；Empty circle: p53.
Fig. 9 is that a kind of compound H109 has the Cre-infection cell inhibitory action of Mdm2 and MdmX to divide to expressing
Analysis.Void column: nutlin-3a (positive control)；Light grey post: nutlin-3b (negative control)；Dark grey post:
Compound H109.A. the Apoptosis assay of the compound H109 induction MEF cell containing Mdm2 and MdmX.
B. the Apoptosis assay of the MEF cell of compound H109 induced deletion MdmX.Such as nutlin-3a, lack
Lose MdmX cell cell quantity after compound H109 processes to significantly reduce.C. same, lack Mdm2 cell
After compound H109 processes, cell quantity also significantly reduces.On the contrary, negative control nutlin-3b is to cell
Vigor does not affect.D. disappearance Mdm2 and MdmX cell is not affected by compound H109.
Figure 10 is the threedimensional model schematic diagram that a kind of compound H109 with N-MdmX is combined.
Detailed description of the invention
Applicant will the present invention is described in further detail in conjunction with specific embodiments below.
Embodiment 1: the preparation method of the micromolecular inhibitor of a kind of MdmX/Mdm2:
The micromolecular inhibitor of MdmX/Mdm2 is cis-imidazolines, its synthesis path as it is shown in figure 1,
Principle is to use to have diastereomeric and stereo selectivity catalyst aromatic nitro methane and the addition of schiff bases
Reaction (Davis, T.A.&Johnston, J.N.Chem.Sci.2,1076-1079,2011).
R1Functional group utilizes nitro-phenyl alkyl (compound 8) to introduce.Using halogeno-benzene alkane is raw material, by Kornblum
Description method prepares (JACS, 1956,78:1497), functional group R1(4) hydroxyl in, R1(5) carboxyl in
And R1(6) amide in is respectively adopted methyl ether, methyl ester and benzyl protection, and post-synthetic phase goes by Hydrolyze method respectively
Protection.
R2Functional group utilizes aryl imine (compound 7) to introduce.Use a-aminobenzene sulfone or a-amino indole sulfone
For raw material, utilize potassium carbonate to carry out eliminating reaction and generate schiff bases (Marianacci et.al., Chem-Euro
J, 2007,13:8338).
R3Functional group uses corresponding benzoic acid derivative to be that synthesis material introduces.Functional group R3Carboxyl in (4),
R3And R (5)3(6) amide in is respectively adopted methyl ester and benzyl protection, and post-synthetic phase goes to protect by Hydrolyze method respectively
Protect.
Through tertbutyloxycarbonyl protection aryl imine (compound 7) and nitro-phenyl alkyl (compound 8) press 1:1.1
Equivalent is dissolved in toluene (0.1M), under trifluoromethayl sulfonic acid (HOTf, 5% mol ratio) catalytic action,
-78 DEG C are reacted 19～27 hours, generate beta-amino nitroparaffin intermediate (compound 9) by additive reaction.
Utilize boronation cobalt that nitro in compound 9 is reduced into amino, it is provided that acylation reaction introduces R3 functional group, generate
The amide compound (compound 10) of tertbutyloxycarbonyl protection.Direct TFA removes t-butoxycarbonyl protecting group
Generating secondary amine, productivity is more than 90%, then amido acylation reaction generates under N, N-carbonyl dimidazoles catalyst
Carbimide. intermediate product, this intermediate product processes generation cis-imidazolines nutlin-3a through piperazinones and is similar to
Thing, whole productivity is 71～89%.
Course of reaction utilizes LC-MS to monitor, until converting completely.Reactant water extracts, and water layer is again
Extract with dichloromethane.Organic layer is merged, is dried with sodium sulfate, is concentrated to give crude product.Use WatersHPLC
Anti-phase C18 post is further purified, and mobile phase A is the water containing 0.1% formic acid, and Mobile phase B is containing 0.1% first
The methanol of acid, Mobile phase B, from 0 to 45% linear gradient elution, is collected main eluting peak, is passed through hands further
Property chromatographic column SFC (OD-H post) separate, obtain purer enantiomer (> 99%).
Embodiment 2:MdmX and Mdm2 protein sample preparation method:
Amino-terminal amino acid 22-110 sequence fragment (N-MdmX) of people source MdmX and Mdm2 aminoterminal amino
Acid 22-110 (N-Mdm2) sequence fragment, by the relevant DNA of escherichia coli preferred codons synthesis, synthetic
DNA fragmentation process stand-by with restricted enzyme BamH I and EcoR I respectively.
Protein expression uses commercialization pET28a plasmid (Novagen), beforehand through site-directed mutagenesis technique by it
Original blood coagulation restriction enzyme site (Leu-Val-Pro-Arg-Gly-Ser) is replaced as the enzyme action position of TEV protease
Point (Glu-Asn-Leu-Tyr-Phe-Gln-Gly), the named pSD of novel plasmid.The identical restricted enzyme of pSD
Process stand-by.
N-Mdm2 with the MdmXDNA fragment processed through BamH I and EcoR I respectively with identical restricted
The pSD carrier that restriction endonuclease processed connects, and builds N-Mdm2 and MdmX protein expressing plasmid, pSD-Mdm2
And pSD-MdmX.Two kinds of newly-built expression plasmids proceed to escherichia coli DH5a competent cell respectively by electric shocking method
Carry out plasmid amplification and DNA sequencing confirms.
The expression plasmid confirmed through DNA sequencing proceeds to e. coli bl21 (DE3) sense respectively by electric shocking method
N-MdmX and N-Mdm2 gene engineering expression bacterium is prepared by state cell.
N-MdmX and N-Mdm2 albumen utilizes said gene engineering expression bacterium to prepare through expression, separation and purification.
Carry out cell with LB culture medium to cultivate to OD600nmWhen=1.0, at 20 DEG C, cultivate 12 with the IPTG of 0.4mM
Hour induced protein is expressed.Centrifugal collecting cell culture (8000g, 30 minutes), cell precipitation is suspended in
In 50mM phosphoric acid buffer buffer, carry out supersound process 4 minutes.High speed centrifugation lysate (100,000 ×
G) centrifugal clear cell debris, supernatant is loaded to the Ni-NTA agarose column (Qiagen) of 20ml column volume
Purification.Collect the component Han target protein, through 4 DEG C of digested overnight of TEV protease, remove the histidine of isolating protein
Label, is then passed through second time Ni-NTA agarose chromatography purification and removes non-digestible protein and free histidine mark
Sign.Eluting collects target protein component, is concentrated to 1 mg/ml, uses solvent resistant column S200 further
Carry out molecular sieve purification.Merge target peak, and be concentrated into 1 mg/ml then with liquid nitrogen flash freezer, and preserve
Standby in-80 DEG C.
With N-MdmX and the N-Mdm2 protein sample of nuclear magnetic resonance experiment, containing BME vitamin (Sigma)
M9 minimal medium in, use 1 grams per liter 15N-ammonium sulfate to be marked as nitrogen source, other step with
Described in before.Detecting through SDS-PAGE electrophoretic analysis, lipidated protein is more than 98%.
Embodiment 3, high flux MdmX inhibitor screening method:
MdmX inhibitor screening uses fluorescence polarization (FP) method to measure.Containing 10mMTris (pH value 8.0),
The mensuration buffer of NaCl and 0.01%Tween-20 of 200mM is carried out.People source p53 Argine Monohydrochloride
15-29 sequence fragment peptide (p53p) with fluorescein be marked (fluorescein-GSGSSQETFSDLWKLLPEN,
Flu-p53p).Mutant p53 peptide (fluorescein-GSGSSQETASDLAKLAPEN, Flu-p53pAAA) is used as
Negative control.
FP detection all uses 15nM fluorescein and the N-MdmX of 1 μM, containing 10mMTris (pH value
8.0), the buffer of NaCl and 0.01%Tween-20 of 200mM measures.Suppress at N-MdmX/p53p
In agent determination test, nutlin analog and albumen are pre-mixed 30 minutes, are subsequently adding labelling peptide and mix
30 minutes.Then carry out (Corning) FP with 384 hole black microwell plates to measure.Use with 555nm
Exciter filter, the static state of 632nm and the EnVision multiple labeling microplate reader of polarizing filters carry out FP test
Analyze.Unlabelled p53p peptide and nutlin-3 α is utilized to make positive control, and alanine substituted p53 peptide
(p53pAAA) it is used as negative control.
The present invention first passes through high throughput method screening lead compound.The present invention aims at design MdmX
High-affinity inhibitor, therefore chooses IC50The value little molecule less than 300nM is as MdmX/Mdm2 first
Lead inhibitor.From the nutlin-3a analog library of design, it has been found that the IC of six kinds of micromolecular compounds50
Value is respectively less than 300nM, and high flux screening result is as shown in Figure 5.These six kinds of micromolecular compounds are respectively
H077, H109, H135, H181, H196 and H206, they chemical structural formulas are as shown in table 2.
Table 2, the chemical structural formula of six kind of guide's inhibitor
Embodiment 4, identical titration calorimetry (ITC) analyze method
At 25 DEG C, utilize the micro-calorimeter of isothermal titration, ITC-200 (GEHealthcare company/MICROCAL)
Carry out protein-ligand interaction to measure.Typical experiment is for by 19 equal portions titration sample introductions the most about 0.2
The ligand solution (each 2.1 microlitres) of mM is to containing in the protein solution ITC calorimetric pond of about 10～20 μMs
(volume is 200 μ L).Contrast test uses buffer titration protein solution.Sky is deducted from experimental data
White experimental data, has carried out analysis and has obtained the thermodynamic parameters such as enthalpy change isothermal line.When necessary, benchmark can be through
Manually regulation, to reduce background noise.Based on the Origin7.0 provided by instrument GE/MICROCAL
Software carry out data analysis and one group of location pattern is used as basic option.Association constant Ka is
(1/Kd) solution is determined by the slope of the centre straight ahead part of saturation curve.Gibbs free energy Δ G and Entropy Changes
Δ S difference: Δ G=-RTlnKa=Δ H-T Δ S formula calculates, and wherein Δ H obtains from ITC titration curve
?.
Make in aforementioned manners the affinity of above-mentioned six kinds of lead compound with N-Mdm2 and N-MdmX to be surveyed
Fixed, binding constant is shown in table 1 in summary of the invention, and wherein compound H109, H181 and H206 is to N-Mdm2's
Affinity is less than 50nM.And, compound H109 is less than 30nM to the binding constant of N-MdmX and N-Mdm2,
It is respectively 27nM and 5.7nM.
The ITC experimental result of N-MdmX and N-Mdm2 is shown in Fig. 6 by compound H109.
Embodiment 5, the dynamic method of nuclear magnetic resonance spectroscopy analyzing proteins skeleton
All NMR spectra utilize the Bruker Avance600 equipped with three nuclear resounce pulsed magnetic field gradients probes
Megahertz spectrogrph gathers 25 DEG C of detections.H1-N15HSQC H NMR spectroscopy is with TPPI type collection.All of NMR
Sample is by containing 20mM sodium phosphate, 200mM sodium chloride, 2mM DTT, 0.02%NaN3, and 95%H2
O/5%D2Prepared by the buffer of O, pH6.0.The ultimate density of albumen composition is about 0.4mM.All numbers
NMRPipe process is used according to collection.Use NMRView software kit to carry out frequency spectrum to show and analysis.Use by method sieve
Pulse train Deng description carries out the mensuration of heteronuclear NOE value, under the conditions of 800 megahertzs, uses 15N labelling
Protein and nutlin-3a complex, carry out the experiment of heteronuclear NOE, the dynamic of analyzing proteins skeleton.
Template compound nutlin-3a (i.e. structural formula compound shown in the middle of Fig. 2, or see Fig. 7) and N-MdmX
H when combining with N-Mdm21-N15HSQC nuclear magnetic resonance map is shown in that the NMR peak of Fig. 3, N-Mdm2 compares N-MdmX
Many, data result display N-Mdm2 with Nutlin3a affinity is stronger than N-MdmX.
Heteronuclear NOE value result when template compound nutlin-3a with N-MdmX, N-Mdm2 are combined is shown in Fig. 4,
Fig. 4 upper part data are to utilize N-MdmX Yu the nutlin-3a mixture (1:1 mol ratio) of 15N labelling,
Fig. 4 lower part data are to utilize N-Mdm2 Yu the nutlin-3a mixture (1:1 mol ratio) of 15N labelling.
The aminoacid sequence 64-97 fragment of result display N-MdmX is flexible stronger than the respective segments in N-Mdm2.This is
One of present invention foundation designing the micromolecular inhibitor of MdmX/Mdm2.
Embodiment 6: micromolecular inhibitor is to cancer cell targeted inhibition effect
The present invention utilizes containing the tumor cell line of wild type p53, including colorectal cancer cell HCT116 and RKO,
Lung carcinoma cell H460a, the activity of test micromolecular inhibitor anticancer.
Three kinds of tumor cells, after Secondary Culture 36～48 hours, are separately added into the chemical combination of 10 μMs (final concentrations)
Thing H109 joins in cell culture medium, mixing, after standing 8 hours, utilizes fresh culture washed cell
Remove medicine 3 to 5 times.Centrifugal collecting cell, broken essence, then centrifuging and taking supernatant is anti-by real time aggregation enzyme chain
Answer the expression of (RT-PCR) monitoring p53 and p21 gene.Result as shown in Figure 8, in all cells system,
Transcribing of p21 gene, consistent with the result of positive control nutlin-3a, all increase in the way of dependent dose.
By contrast, transcribing of p53 gene itself is not affected by compound H109 process.These data show,
Compound H109 raises p53 vigor by mechanism after translation, similar to positive control nutlin-3a effect.
The targeting of cell and growth viability are suppressed by the present invention further by mtt assay analysis of compounds H109
Effect.Mouse model is utilized to prepare Mdm2 deficiency, MdmX deficiency lung cancer in mice embryo fibroblast (MEF),
MEF is from the derivative mice of the MdmX+/-P53-/-mice of 13.5 days and MdmX+/-P53+/-mice intermolecular hybrid
Embryo separates.Native tumoral cell divides from MdmX-/-p53-/-mice or primary lung cancer p53-/-mice
From, gene type is identified by PCR reaction.The MdmX mouse model used, passes through conventional gene
Prize law obtains, and the expression of MdmX is confirmed by RT-polymerase chain reaction, does not the most produce MdmX albumen.
All cells 5%CO2, adding 10% serum and penicillin, the DMEM culture medium of streptomycin is 37 DEG C of cultivations.
In proliferation test, plating cells density is 1x104/cm2, cell culture is divided into 300000 cell/ml,
Then by the cell suspending liquid of 100 μ l, in triplicate, add in transparent whites 96 orifice plate (Corning),
After 2 hours, add MdmX inhibitor or nutlin-3a (sun that 1 microlitre is dissolved in the present invention of dimethyl sulfoxide
Property comparison) or nutlin-3b (negative control, structural formula is shown in Fig. 7), continue to cultivate 48 hours at 37 DEG C.
This flat board adds 100 μ l Cell Titer Glo reagent (Promega) after being down to room temperature, be shaken to mixed 2 minutes,
Then lucifuge is placed 15 minutes, reads cell number (Perkin with Envision2103 multiple labeling plate reader
Elmer), data acquisition MICROCAL Origin software carries out processing (V8.1, MICROCAL).
Analysis result is shown in Fig. 9, and result display non-totally ordered MEF cell is after compound H109 processes 48 hours
Vigor substantially reduces (Fig. 9 a), the same with positive control nutlin-3a, presents dose dependent.Such as Fig. 9 b
Shown in, MdmX deficiency MEF cell also presents dose dependent after compound H109 processes 48 hours and becomes
Change.It is essential that Mdm2 deficiency MEF cell presents dose dependent after compound H109 processes equally
Change (Fig. 9 c).On the contrary, the double defect MEF cell of Mdm2 and MdmX is not the most by compound H109
Impact (Fig. 9 d).These results indicate that nutlin analog institute of the present invention mediated cell viability
Reduction is the vigor strictly relying on p53.
Claims (8)
1. chemical structural formula is
Compound preparation as the purposes in the micromolecular inhibitor medicine of MdmX/Mdm2, wherein, R1
For phenyl, 2,4-Dichlorobenzene base, 4-hydroxy phenyl or 4-carboxyl phenyl；
R2It is 3,5-Dichlorobenzene base, 3,4,5-trichlorophenyl or 5-chloro-indole base or indyl；
R3For 2-(1,1,1-trimethyl) methoxyl group-4-cyano-phenyl, 2-tert-butoxy-4-(3-hydroxyl-3-
Oxo-propionyl) phenyl, 2-tert-butoxy-4-(3-amino-3-oxo-propionyl) phenyl or 2-(1,1,
1-trifluoromethoxy)-4-(3-amino-3-oxo-propionyl) phenyl.
Purposes the most according to claim 1, it is characterised in that: described compound chemical structure formula is
Purposes the most according to claim 1, it is characterised in that: described compound chemical structure formula is
Purposes the most according to claim 1, it is characterised in that: described compound chemical structure formula is
Purposes the most according to claim 1, it is characterised in that: described compound chemical structure formula is
Purposes the most according to claim 1, it is characterised in that: described compound chemical structure formula is
Purposes the most according to claim 1, it is characterised in that: described compound chemical structure formula is
8. according to described purposes arbitrary in claim 1-7, it is characterised in that: described MdmX/Mdm2
Micromolecular inhibitor medicine be anticancer Mdm2/MdmX, discharge repressed wild type p53 egg
In vain, the medicine of inducing cell oneself apoptosis.
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